Abstract The overarching hypothesis for this proposal is that tissue transglutaminase (TG2), a protein we found to be up-regulated in ovarian cancer (OC) regulates epithelial to mesenchymal transition (EMT) in OC cells and the survival of ovarian cancer stem cells by enhancing 2-catenin cellular signaling. TG2, an enzyme overexpressed in several epithelial malignancies, induces Cadependent protein post-translational modifications and cross-linking. We reported for the first time that TG2 is up-regulated in OC and work in our laboratory during the current funding period demonstrated that the enzyme critically regulates the process of OC metastasis. We showed that when injected intraperitoneally or under the ovarian bursa of nude mice, OC cells engineered to express decreased levels of TG2 induced significantly less peritoneal dissemination compared to control cells. We also showed that TG2 induces EMT of OC cells and regulates formation of spheroids from OC cells. These discoveries led us to formulate the hypothesis that TG2 is critical to the process of EMT and the survival of ovarian cancer stem cells, which we will test by addressing three objectives: Aim 1: Identify the mechanisms by which TG2 regulates 2-catenin stability and study its effects on 2-catenin dependent gene transcription. We will study whether TG2 knockdown or inhibition alters 2-catenin cellular localization, phosphorylation and signaling in response to Wnt signals, contributing to EMT of OC cells. Aim 2: Determine whether TG2 induced EMT and metastasis are regulated by 2-catenin signaling. We will investigate whether 2-catenin knock-down inhibits TG2 induced-EMT and metastasis and whether its stabilization induces EMT in cells expressing low levels of TG2. Aim 3: Establish the effects of TG2 expression on the survival and functions of ovarian cancer stem cells. We will measure and compare TG2 expression in ovarian CSCs vs. whole tumors. We will knock-down TG2 or inhibit its enzymatic activity to block the functions of ovarian CSCs (e.g. survival, formation of spheroids and tumorigenicity). Significance: We are the first group studying the role of TG2 in ovarian cancer focusing on mechanisms of metastasis and EMT modulated by TG2. This proposal is timely and highly relevant as EMT has been recently linked to the cancer-stem cell phenotype. Our group has produced evidence for the presence of such a distinct population of cells in ovarian tumors. It is likely that persistence of these cells after standard treatment is responsible for the failure of chemotherapy to eliminate cancer. Therefore, demonstrating that TG2 facilitates the survival of ovarian CSCs is highly relevant, as it will identify a new pathway that can be targeted with the goal of eradicating treatment resistant stem cells and of blocking OC metastasis.